Stub1负性调节Treg细胞抑制性功能在重症急性胰腺炎肠粘膜损伤中的作用及机制研究

Latest studies indicate that, immune cells play an important role in the pathophysiological process of SAP and may mediate injuries of the pancreas and extrapancreatic organs. Meanwhile, studies in colitis models suggest that Treg cells play a key role in maintaining the homeostasis of the gut. Our previous studies have revealed that: intestinal Foxp3+Treg cells were markedly decreased in the rat models of SAP and were negatively correlated with the intestinal inflammatory responses. Whereas, the expressions of intestinal Stub1 were obviously up-regulated in SAP rats. Therefore, we hypothesized that SAP would promote the degradation of Foxp3 via activating Stub1 to negatively regulate the intestinal Treg cells, result in the aggravation of intestinal mucosal injury. In this subject, we will use in vivo and in vitro SAP models to: ① identify the changes in numbers and functions of the intestinal Treg cells, ② confirm the protective effect of intestinal Treg cells on the intestinal epithelial cells, ③ investigate the molecular mechanism of the negative regulatory effects of intestinal Treg cells via Stub1. The results of this subject will further enrich the pathogenesis of SAP-induced intestinal injury and provide novel theoretical basis and therapeutic direction for the treatment of SAP.

最新研究表明，免疫细胞在SAP病理生理过程中扮演了重要角色，并可能介导了胰腺及胰外器官的损伤。同时，在结肠炎等动物模型中发现，肠道Treg细胞在维持肠道内稳态方面发挥了关键作用。我们前期研究发现：SAP大鼠肠道Foxp3+Treg细胞显著减少，并且与肠道炎症水平存在明显的负性相关，而肠道Stub1表达明显增加。由此，我们假设：SAP会通过激活Stub1来促进Foxp3的降解，负性调节肠道Treg细胞，加重肠道粘膜损伤。本课题将应用体内及体外SAP模型：①明确SAP时肠道Treg细胞的数量及功能变化；②明确肠道Treg细胞对肠道上皮细胞的保护作用；③明确Stub1是SAP时负性调节肠道Treg细胞的机制。研究结果将进一步丰富SAP时肠道损伤的发病机理，为SAP的治疗提供新的理论依据和治疗方向。

肠道是多器官功能障碍的始动器官，SAP患者往往会合并肠道功能损伤，而肠道功能障碍会进一步加重患者病情，导致恶性循环。我们选取8-10周龄C57BL/6背景的WT小鼠为实验对象，用雨蛙素+LPS建立SAP动物模型，结果显示，SAP造模后会导致明显的肠道损伤，表现为肠道紧密连接蛋白表达的减少、肠道上皮细胞凋亡数量的增加，同时伴有肠道免疫应答的紊乱，主要表现为肠系膜淋巴结Treg细胞的减少以及Th17细胞的增加，Treg细胞向Th17细胞偏移和转化。为了进一步验证Treg细胞在SAP全身重症化中的作用，我们应用WT小鼠和全身性Foxp3 KD小鼠来进行SAP造模，进行相关的体内和体外实验验证。动物实验表明，Foxp3+/-会明显加重SAP的肠粘膜损伤，表现为肠道紧密连接蛋白表达的进一步下降以及肠道上皮细胞凋亡数量的明显增加，同时肠道免疫应答紊乱更为明显，Treg细胞过度地向Th17细胞偏移和转化，同时全身炎症反应以及胰腺损伤更加明显，表现为外周血脂肪酶、淀粉酶以及炎症因子水平的显著升高。体外试验证实，将全身性Foxp3 KD小鼠的肠系膜淋巴结提取的Treg细胞与小鼠小肠粘膜上皮细胞Mode-K细胞共培养后进行LPS刺激，Mode-K细胞的肠道紧密连接蛋白的表达显著降低、炎症反应明显加重，同时凋亡细胞的数量显著增加。以上结果均提示，Foxp3在维持Treg细胞稳定性和抑炎性质方面发挥重要作用，Foxp3的缺失会加重Treg细胞的促炎属性，进而导致肠道炎症反应增加、肠粘膜损伤加重，说明Foxp3+Treg细胞在维持肠粘膜完整及抑制肠道炎症反应方面发挥关键作用，这一发现有望于为临床治疗提供新的治疗思路及治疗方向。